Shan et al. BMC Genomics (2020) 21:49 https://doi.org/10.1186/s12864-020-6454-y RESEARCH ARTICLE Open Access Transcriptome analysis of Clinopodium gracile (Benth.) Matsum and identification of genes related to Triterpenoid Saponin biosynthesis Chunmiao Shan1,2†, Chenkai Wang1,2†, Shengxiang Zhang1,2, Yuanyuan Shi1,2, Kelong Ma1,3, Qingshan Yang1 and Jiawen Wu1,2,4* Abstract Background: Clinopodium gracile (Benth.) Matsum (C. gracile) is an annual herb with pharmacological properties effective in the treatment of various diseases, including hepatic carcinoma. Triterpenoid saponins are crucial bioactive compounds in C. gracile. However, the molecular understanding of the triterpenoid saponin biosynthesis pathway remains unclear. Results: In this study, we performed RNA sequencing (RNA-Seq) analysis of the flowers, leaves, roots, and stems of C. gracile plants using the BGISEQ-500 platform. The assembly of transcripts from all four types of tissues generated 128,856 unigenes, of which 99,020 were mapped to several public databases for functional annotation. Differentially expressed genes (DEGs) were identified via the comparison of gene expression levels between leaves and other tissues (flowers, roots, and stems). Multiple genes encoding pivotal enzymes, such as squalene synthase (SS), or transcription factors (TFs) related to triterpenoid saponin biosynthesis were identified and further analyzed. The expression levels of unigenes encoding important enzymes were verified by quantitative real-time PCR (qRT-PCR). Different chemical constituents of triterpenoid saponins were identified by Ultra-Performance Liquid Chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS). Conclusions: Our results greatly extend the public transcriptome dataset of C. gracile and provide valuable information for the identification of candidate genes involved in the biosynthesis of triterpenoid saponins and other important secondary metabolites. Keywords: Clinopodium gracile (Benth.) Matsum, Transcriptome, RNA-Seq, Triterpenoid saponin biosynthesis, Differentially expressed genes Background East Asia [3]. According to the results of previous stud- Clinopodium gracile (Benth.) Matsum (C. gracile), ies, triterpenoid saponins in C. gracile exhibit several known as the tower flower, is a traditional Chinese herb, pharmacological effects, as these possess anti- which belongs to Lamiaceae and grows on wasteland, inflammatory [4], anti-hepatoma [4, 5], cardioprotective roadsides, and hillsides [1, 2]. Approximately 20 Clino- [6], anti-tumor [7], and immunoregulatory [8] proper- podium species are found in Europe, Central Asia, and ties. Triterpenoid saponins were synthesized from two C5 isoprene units, isopentenyl pyrophosphate (IPP) and * Correspondence: [email protected] dimethylallyl diphosphate (DMAPP). These components †Chunmiao Shan and Chenkai Wang contributed equally to this work. were condensed in a sequential manner by prenyltrans- 1 Anhui University of Chinese Medicine and Anhui Academy of Chinese ferases, resulting in the formation of prenyl diphos- Medicine, Hefei 230038, China 2Key Laboratory of Xin’an Medicine, Ministry of Education, Anhui University phates, such as geranyl pyrophosphate (GPP) and of Chinese Medicine, Hefei 230038, China farnesyl pyrophosphate (FPP), which are further Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shan et al. BMC Genomics (2020) 21:49 Page 2 of 16 transformed into the carbocyclic skeleton of triterpenoid tissues were statistically significant (Fig. 1, Add- saponins by the action of squalene synthase (SS) and itional file 1: Table S1). squalene epoxidase (SE). Finally, the backbone is chem- ically modified by cytochrome P450 monooxygenase Analysis of triterpenoid saponins in C. gracile by UPLC/Q- (CYP450) and UDP-glycosyltransferase (UGT), resulting TOF-MS in the production of different types of triterpenoid sapo- Qualitative analysis of triterpenoid saponin metabolites nins [9–12]. However, genes encoding key triterpenoid of C. gracile was conducted by UPLC/Q-TOF-MS. The saponin biosynthesis enzymes in C. gracile are largely results confirmed the presence of Buddlejasaponin IV in unknown. C. gracile. Additionally, Saikosaponin a, Clinoposaponin Squalene synthase (SS; EC 2.5.1.21) is a key bifunc- III, and Clinoposaponin V probably also exist in C. gra- tional enzyme in the terpenoid biosynthesis pathway; SS cile, according to the retention time (tR), maximum first catalyzes the formation of presqualene diphosphate ultraviolet absorption wavelength (λmax), molecular ion (PSPP) from two molecules of FPP and then converts peak, and ESI-MS data (Fig. 2, Additional file 2: Table PSPP to squalene with NADPH and Mg2+ ions [13, 14]. S2). The level of SS gene expression shows a positive correl- ation with the amount of triterpenes [15]. Although SS RNA sequencing and the de novo assembly of C. gracile cDNAs have been cloned and analyzed from a number transcriptome of herbal plant species, such as Glycyrrhiza glabra [16], RNA-Seq analysis of C. gracile flowers, leaves, stems, Siraitia grosvenorii [17], and Lotus japonicas [18], no se- and roots generated 43.81 Gb of high quality reads. Full- quence or structural information is available on the SS length transcripts were reconstructed, and a total of 128, gene in C. gracile. 856 unigenes, which refer to a uniquely assembled tran- RNA sequencing (RNA-Seq) analysis has been used to script or a cluster of genes that perform a particular capture both coding and non-coding sequences and to function, were generated using Trinity and TGICL, with quantify gene expression not only in a heterogeneous a mean length of 1354 bp and an N50 value of 2268 bp. mixture of cells, tissues, and organs but also at the whole Of the 128,856 unigenes, 47.37% (61,043 unigenes) were organism level [19]. Thus, RNA-Seq is a powerful tool longer than 1000 bp, and 34.87% (44,929 unigenes) were for acquiring gene expression information and is of great longer than 1500 bp (Additional file 3: Figure S1). significance in the mining of functional genes, analysis of gene expression profiles, and discovery of genetic Functional classification and expressed overview of metabolic networks [20]. At present, transcriptome se- unigenes quencing has been applied to medicinal plants of the Of the 128,856 unigenes, 38.99, 57.16, 58.94, 72.57, Labiatae family, including Scutellaria baicalensis Georgi 54.23, 54.96 and 55.67% mapped to GO, KEGG, KOG, [21], Ocimum sanctum and Ocimum basilicum [22], NR, NT, Pfam, and SwissProt databases, respectively. Mentha piperita and Mentha arvensis [23], and Clinopo- Additionally, 99,020 (76.85%) unigenes were annotated dium chinense [24]; however, the use of RNA-Seq ana- lysis has not been reported in C. gracile to date. In this study, we performed deep transcriptome ana- lysis of four different tissues (flowers, leaves, roots, and stems) of C. gracile plants and identified genes poten- tially involved in triterpenoid saponin biosynthesis. This work lays a foundation for further exploration of the molecular mechanism of triterpenoid saponin biosyn- thesis in C. gracile. Results Total saponin content in different tissues of C. gracile Saponins were extracted from approximately 0.1 g dried powder of flowers, leaves, stems, and roots of C. gracile. Total saponin content was the highest in leaves (0.29%), followed by stems (0.23%), flowers (0.21%), and roots Fig. 1 The total saponin content of leaves, stems, flowers, and roots (0.18%), with a standard error of 0.0005, 0.0004, 0.0033, of Clinopodium gracile (Benth.) Matsum plants. The X-axis indicates and 0.0011, respectively. The results of analysis of vari- the type of C. gracile tissues, and the Y-axis indicates the total ance (ANOVA) of C. gracile data showed that the differ- saponin content. Significant differences were determined by analysis of variance (ANOVA) ences among the total saponin content of the four Shan et al. BMC Genomics (2020) 21:49 Page 3 of 16 Fig. 2 MS/MS spectra of Saikosaponin a (a), Buddlejasaponin IV (b), Clinoposaponin III (c) and Clinoposaponin V (d)ofC. gracile by UPLC/Q-TOF- MS in negative ion mode in at least one public database, while a total of 25,975 GO terms within the “cellular component” category; (20.16%) unigenes were co-annotated in five databases “catalytic activity” (24,182 unigenes [43.45%]), and “bind- (Table 1). Species distribution analysis showed that C. ing” (24,147 unigenes [43.48%]) were the most enriched gracile unigenes showed the highest homology to Ses- GO terms under “molecular function”; “cellular process” amum indicum sequences (40,961 unigenes; 43.81%), (13,531 unigenes [43.45%]), and “biological regulation” followed by Erythranthe guttata (14,690 unigenes; (4852 unigenes [15.58%]) were the most enriched GO 15.71%) and Daucus carota subsp. sativus (8866 uni- terms under “biological process” (Additional file 5: Fig-